The invention relates to a method and an apparatus for carrying out that method for the simultaneous specific and quantitative determination of several substances capable of immunoreaction. The substances are especially of the kind capable of effective antigen action and the sample migrates from an antibody-free carrier into a carrier containing further constituents which, together with the substances to be determined, generate an immunoreaction. The substances which are to be analyzed form precipitates and the extent of migration of the sample takes place in the direction of an electric field which extends over the sample and over the carrier. An apparatus for carrying out this method, as well as advantageous uses of the method, are also part of the invention.
An important field of application of a method such as described above is, for example, the quantitative determination of proteins in blood plasma. Such plasma proteins are for example: Prealbumin, Albumin, .alpha.-Lipoprotein, .alpha.-I-Antitrypsin, .alpha.-I-B-Glykoprotein, Gc-Globulin, Coeruloplasmin, .alpha.-2-Macroglobulin, Gc-Globulin, Coeruloplasmin, .alpha.-2-Macroglobulin, Pseudocholinesterase, hemopexin, Transferrin, .beta.-Lipoprotein, Haptoglobin, Orosomucoid, Antitrypsin, C-reactive protein, Fibrinogen, Plasminogen, IgG, IgM, IgA, IgD. The quantitative determination of these proteins in many cases permits differential diagnostic conclusions to be made or else, when the diagnosis has been made, it yields further information regarding the progress and prognosis of a disease (H. H. Marki, Analytische Methoden zur Darstellung der Serumeiweisskorper und ihre Aussagemoglichkeiten fur Klinik und Praxis, Deutsches Medizinisches Journal, 1972 (Jg. 23), S. 317 ff.; H. J. Braun, Immunglobuline, Paraproteine und Blutfarbstoff bindende Proteine und ihre Bedeutung fur die Klinik, Deutsches Medizinisches Journal, 1972 (Jg. 23) S. 227 ff.; C. O. Kindmark + C. B. Laurell, "Sequential Changes of the Plasma Protein Pattern in Inoculation Hepatitis," Scand. J. clin. Lab. Invest. 29, suppl. 24, 105-115 (1972); J. S., Hepatitis: IgA-Mangel erhoht Risiko, SELECTA 41, S. 3574 (1974); as well as the research of R. Scherer, A. Moratescu, and G. Ruhenstroth-Bauer, "Die spezifische Wirkung der Plasma-Proteine bei der Blutkerperchensenkung").
Several methods have been published for the quantitative determination of single substances (antigens) which produce immunoreaction with antibodies or, again, methods for the quantitative determination of substances (antibodies) which produce immunoreaction with antigens. Such methods are disclosed for example in W. Becker, B. Rapp, H. G. Schwick und K. Storiko, "Methoden zur quantitativen Bestimmung von Plasmaproteinen durch Immunprazipitation," Zeitschrift fur klinische Chemie und klinische Biochemie, 1968, Heft 3, S. 113-122; W. Becker, "Methoden der qualitativen und quantitativen Immun-Elektrophorese," Hrsg. Behringwerke AG, Frankfurt, 1972; Prospekt "DAKO-Immunoglobulins" der DAKOPATTS A/S., Danemark, 1972. All these methods are based on the principle of immunodiffusion and/or electrophoresis.
When the concentration of antigens is to be determined by the method of immunodiffusion, the antigens contained within a sample diffuse into a carrier, for example a layer of agarose gel. The carrier contains only a particular antibody specific to a particular antigen. When antigens and antibodies make contact within the carrier, they react and form a complex which is precipitated within the carrier. The diffusion process proceeds until the specific antigen within the sample has been entirely used up by precipitation with the specific antibody in the carrier. Any remaining antigens within the sample can continue to diffuse into the carrier without hindrance and they form no precipitate. The area within the carrier in which a precipitate is formed can be made visible by staining: for example in the so-called radial immunodiffusion process, this area is circular, i.e., it forms a circular zone around the application point at which the sample was applied to the carrier. The dimension of this area in which a precipitate was formed is thus a measure for the concentration of that antigen within the sample against which the antibody contained within the carrier was specifically directed.
In principle, this method may be used for a quantitative determination of all such antigens against which specific antibodies are known or against which specific antibodies can be produced. However, this method is very time-consuming and does not permit the simultaneous determination of several antigens.
The above-described method may be accelerated by the use of electrophoresis. Use is made of the fact that, under certain ambient conditions, antigens carry an electric charge. If an electric field is applied in the vicinity of the carrier, the electrically charged antigens pass through a substantially larger path within the carrier than would be the case in pure diffusion.
In the general practice, several methods for the determination of the concentration of individual antigens are used:
a. In the so-called acetate foil electrophoresis a sample (generally human serum) is split up, by an electric field adjacent to the foil, according to the different migration velocities of the individually contained components (antigens). After the separation, the sample is stained and is evaluated photometrically. The separation process normally used in clinical practice only determines a few groups, each of which has the same electrophoretic migration characteristics. Thus the differentiation is confined to a determination of .alpha., .beta., .gamma.-Globulin and Albumin. Any quantification within these groups, i.e., the determination of the individual antigens (proteins) contained within each group is not possible by this method.
b. In the process of radial immunodiffusion, the antigens within a sample diffuse from a cylindrical starting orifice in the radial direction into a layer of agarose gel of uniform thickness which contains antibodies. Thus, a cylindrical precipitate is formed around the starting orifice. When the concentration of antibodies in the layer of agarose gel is known, the circular area of the precipitate at the termination of the diffusion process is a measure of the quantity of antigen contained in the sample.
This method normally employs immunodiffusion plates having several starting orifices. In order to obtain a reference curve, several of these starting orifices are filled with different concentrations of a standard solution of known concentration and containing a particular antigen, namely that antigen against which the carrier contains antibodies. The remaining starting orifices are filled with different samples whose content of the same antigen is to be determined. When the diffusion process is complete, (after approximately 48 to 72 hours) the areas of the precipitates are measured and the measured values of the different concentrations of the standard solution represent a reference curve. This reference curve is used to determine the concentration of the particular antigens in the sample against which the antibody in the carrier is directed.
A disadvantage of this method, is first of all, the relatively long duration of the complete formation of the precipitate, i.e., 48 to 72 hours. Furthermore, this method is capable of measuring only the concentration of a single specific antigen at one time. If the concentration of several specific antigens is to be determined, the above-described process must be repeated for each antigen. This method is thus very time and labor consuming.
c. In the so-called rocket immuno-electrophoresis, the disadvantage of great time consumption can be avoided in that the immuno-precipitation is accelerated by the application of an electric field. Just as in the radial immunodiffusion process, individual orifices in a plate, which has a surface carrier of agarose gel, are filled with different concentrations of a standard preparation so as to obtain a reference curve, and several samples with unknown content of a particular antigen are then introduced. After the application of an electric field, the sample migrates from the starting orifices into the carrier which contains antibodies that are specific against the antigen whose concentration is to be measured. For example, if the sample is serum, the antibodies would be specific against a serum protein. the migration of the proteins in the electric field results in precipitates which resemble "rockets" whose height is a measure of the concentration of those antigens within the sample which have undergone an immunoreaction with the antibodies contained in the carrier.
It is a disadvantage of this process that, just as in radial immunodiffusion, there is no possibility for the simultaneous determination of several antigens within a probe because the antibodies in the carrier which form the immunoreaction with the antigens are necessarily specific for only a particular antigen in the sample. In order to determine the concentration of, for example, ten different antigens within a sample (for example, 10 different plasma proteins in a blood plasma of a patient) 10 different carriers with different antibodies of the above-described type must be provided with samples and must be kept in electrophoretic chambers under electric current and subsequently individually evaluated. Until the present time, the great amount of instrumentation and apparatus, as well as the cost of labor, has prevented a wide use of this method in clinical practice.
d. In the process of line immuno-electrophoresis (J. Kroll, Line Immunoelectrophoresis, in: M. H. Axelsen et al.: A Manual of Quantitative Immunoelectrophoresis, Oslo 1973, S. 61-67) a strip of gel which contains the sample with different antigens is contacted to several gel strips, each of which contains polyvalent antisera. After electrophoresis, the immunoreaction of the different antigens in the sample with the several antibodies contained in the polyvalent antisera form precipitate lines in each of the gel strips containing antisera. Since the gel strips containing the several polyvalent antisera are adjacent to one another, the precipitate lines in one strip extend into those of the neighboring strip and thus permit the comparison of the line spectra of the different polyvalent antisera.
e. A known method of the above-described type, on which the present invention is based, is the so-called two-dimensional immuno-electrophoresis according to Clarke and Freeman. This method permits the simultaneous specific (qualitative) and quantitative determination of several antigens, i.e., for example several plasma proteins in blood plasma. In this method, the individual antigens (plasma proteins) are placed in a starting orifice in an agarose gel containing no antibodies and are separated purely electrophoretically in a particular direction (first dimension). Subsequently, the electric field is applied in a second direction (second dimension) which is perpendicular to the first direction. Thus, the once separated antigens now travel in the direction of the second dimension into a carrier which contains antibodies against the various antigens. Thus, in the second dimension, there takes place the same process of electro-immunoprecipitation with the formation of bell-shaped precipitates which overlap. As before, the area of precipitation is a measure of the concentration of the particular antigen (plasma protein) in the applied sample. It is not sufficient however, as in rocket electrophoresis, merely to measure the height; rather the area must be measured and this may be done either by planimetry or after transfer of the outline to paper of normalized thickness by cutting out the figure and weighing it.
The disadvantage of this known method is the extremely great time consumption associated with an evaluation of the precipitates. A further disadvantage is that it is extremely difficult to make a correct interpretation of the resulting, very complicated appearance of the electrophoretic picture, i.e., to associate the different areas of precipitate with the correct antigens (plasma proteins). This is extremely difficult and requires a great deal of experience, particularly when the electrophoretic picture is substantially different from the expected normal case, as will be true in serum samples taken from hospital patients, due to possible pathology.
In summary, the state of the art is such that the determination of several substances capable of an immunoreaction is extremely complicated, expensive and time consuming. The methods a, b, c, described above do not permit the simultaneous determination of several substances within a sample. The method d does permit simultaneous determination but, as has already been mentioned, has the disadvantage that the formation of the precipitate areas to be evaluated takes a very long time and that the evaluation of the precipitates involves a number of very difficult questions of interpretation and identification, particularly in pathologicaly altered patient sera, while the required determination of the precipitate areas, either by measurement of by weighing, is extremely circuitous and time-consuming. It is these disadvantages which are the primary reason that the differential diagnostic methods based on the quantitative determination of several defined antigens, which are available in preliminary form, have been used until the present time only in highly specialized laboratories and require specially trained operators.